A humanized chemogenetic system inhibits murine pain-related behavior and hyperactivity in human sensory neurons

Hyperexcitability in sensory neurons is known to underlie many of the maladaptive changes associated with persistent pain. Chemogenetics has shown promise as a means to suppress such excitability, yet chemogenetic approaches suitable for human applications are needed. PSAM4-GlyR is a modular system based on the human α7 nicotinic acetylcholine and glycine receptors, which responds to inert chemical ligands and the clinically approved drug, varenicline. Here, we demonstrated the efficacy of this channel in silencing both mouse and human sensory neurons by the activation of large shunting conductances after agonist administration. Virally mediated expression of PSAM4-GlyR in mouse sensory neurons produced behavioral hyposensitivity upon agonist administration, which was recovered upon agonist washout. Importantly, stable expression of the channel led to similar reversible suppression of pain related behaviour even after 10 months of viral delivery. Mechanical and spontaneous pain readouts were also ameliorated by PSAM4-GlyR activation in acute and joint pain inflammation mouse models. Furthermore, suppression of mechanical hypersensitivity generated by a spared nerve injury model of neuropathic pain was also observed upon activation of the channel. Effective silencing of behavioural hypersensitivity was reproduced in a human model of hyperexcitability and clinical pain: PSAM4-GlyR activation decreased the excitability of human induced pluripotent stem-cell-derived sensory neurons and spontaneous activity due to a gain of function NaV1.7 mutation causing inherited erythromelalgia. Our results demonstrate the contribution of sensory neuron hyperexcitability to neuropathic pain and the translational potential of an effective, stable and reversible humanized chemogenetic system for the treatment of pain.


Vector generation
To generate our insert, (mCherry-Tandem-PSAM4-GlyR), four fragments were generated (Table S1), digested using restriction enzymes (NEB) and ligated using Quick ligase (NEB); After fragment ligation, the product (2.2kb), was PCR amplified and inserted into a pAAV-CAG backbone using NheI and AgeI restriction sites.From this construct, we next generated our pAAV-CAG-mCherry control plasmid.We PCR amplified NheI-Kozak-mCherry, and inserted it into our pAAV-CAG backbone using NheI and AgeI restriction sites.All constructs were screened and sequence confirmed using Sanger sequencing.

Human embryonic kidney 293T cells and transfection
HEK293T cells were routinely cultured in Dulbecco's modified Eagle Medium (DMEM, Thermofisher scientific) and 10% foetal calf serum.Cells were periodically split using Versene solution (Gibco) and mechanical dissociation.Dissociated cells were seeded into 6 well plates and when cells reached 70% confluence, were transfected using JetPEI following the manufacturer's protocol (PolyPlus transfection).A total of 3 μg of DNA/per 35 cm well was combined with NaCl and JetPEI and after 20 mins added to HEK293T cells.The next day transfected cells were re-plated onto cover slips and used 24 hrs later.

DRG neuron culture and electroporation
Briefly, mice were sacrificed and spinal columns removed.Dorsal root ganglia were rapidly dissected and enzymatically digested at 37°C for 60-90 mins in dispase type II (4.7 mg/ml) and collagenase type II (4 mg/ml).Cells were briefly centrifuged and HBSS/CollagenaseDispase removed.Pre-warmed culture media (Neurobasal, 2% B-27 supplement, 1% Penicillin streptomyocin) was added and cells were mechanically dissociated using fire-polished pipettes.Neurons were transfected via electroporation using the Neon system (Life technologies).Dissociated cells were re-suspended in 10 μl of Buffer R plus 1 μg of total plasmid DNA per 50-100,000 cells.The electrical protocol applied was three 1500-V pulses of 10 ms duration.Cells were immediately plated on Poly-D-lysine/Laminin coated cover slips with the addition of growth factors (mouse nerve growth factor (50 ng/ml; NGF, PeproTech) and 10 ng/ml glial-derived neurotrophic factor (GDNF, PeproTech)).Cells were used for further experiments up until day 4 in vitro.

Generation and culture of induced pluripotent stem cells
Healthy control iPSCs, AD2-1 and AD3-1 (StemBANCC Consortium), were derived from fibroblasts as described previously (81).Data obtained AD2-1 and AD3-1 hiPSCs were pooled and used as control.Another line, RCi002-A, was derived from a patient with inherited erythromelalgia and carries the F1449V mutation in SCN9A (EBiSC Consortium) (44).All lines were separately reprogrammed by non-integrating Sendai viral vectors using the CytoTune-iPS Reprogramming Kit (ThermoFisher).For quality control all iPSC lines were subject to strict checks before initiation of differentiation, including; tests for Sendai virus clearance, FACS for pluripotency markers, genomic integrity checks, cytoSNP analysis for copy number variation and embryoid body tri-lineage differentiation experiments.Cells are also confirmed as negative for Mycoplasma before cryopreservation.iPSCs were maintained in mTesR1 (StemCell Technologies) or StemFlex (Life Technologies) on Matrigel (Corning) coated dishes.Cells were routinely passaged at 80% confluence with EDTA (Life Technologies).Medium was supplemented with Y-27632 (Tocris) when thawing iPSCs.
The base medium was gradually transitioned to N2/B27 medium (Neurobasal medium, 2% B27 supplement, 1% N2 supplement, 1% Glutamax, (ThermoFisher)) in 25% increments.Cells were replated onto glass coverslips at day 12 of the differentiation in N2/B27 medium supplemented with four recombinant growth factors at 25ng/ml (BDNF; ThermoFisher, NT3, NGF, GDNF; Peprotech).CHIR90221 was included for 4 further days.Medium changes were performed twice weekly after replating onto coverslips.If required, Cytosine b-D-arabinofuranoside (araC, 1-2 mM, Sigma) was included in the medium soon after replating to kill the few non-neuronal dividing cells remaining in the culture.AraC was withdrawn from the medium once a pure neuronal culture was obtained, as judged by the absence of morphologically non-neuronal cells on phase-contrast light microscopy.This state was typically achieved 2-3 weeks after replating.From day 28, the concentration of all four recombinant growth factors was reduced to 10ng/ml.Phenol-free Matrigel (Corning, 1:500 dilution) was included in all medium changes from day 28 onward.Medium changes were performed twice weekly.AAVs (AAV9-CAG-mCherry-T-PSAM 4 -GlyR, multiplicity of infection (MOI): 1M, and AAV9-CAG-mCherry, MOI: 100K) were added to the cultures around day 50.AAVs remained in culture for 7 days without media change.Biweekly media changes resumed thereafter.Cells were used for experiments at least 4-6 weeks post AAV infection.

Intrathecal infusion
Each animal was anaesthetised using 2% isoflurane and prepared for surgery by shaving a region over the thoracic vertebrae.T-10 and T-11 vertebrae were located, an incision was made followed by removal of soft tissue to expose the dura and spinal cord.A drop of lidocaine was applied to the dura for approximately 1-2 mins then removed.Using a 30 gauge needle the dura was carefully punctured (CSF leak at this point suggested a successful puncture).A cannula system was designed by connecting tubing of decreasing size until the final cannula tip measured 0.008 in (O.D) x 0.004 in (I.D).The end of the cannula was inserted approximately 1 cm caudal into the subdural space.Using a syringe pump driver, 8 μl of AAV was injected into the subdural space at a rate of 1 μl/min.Following injection, the cannula was allowed to rest in position for 2 min before being slowly removed.The dura was coated with a single drop of dura gel (Cambridge NeuroCare) to seal the dura and prevent further CSF leak.Finally, the incision site was sutured closed and appropriate post-operative care and analgesics given (local 2 mg/kg Marcain, AstraZeneca and systemic 5 mg/kg Rimadyl, Pfizer).Animals were used for behaviour or histology at least 6 weeks post-surgery.

Intra-articular injection
Intra-articular injections of AAVPHP.s-eGFP or AAVPHP.s-mCherry-T-PSAM 4 -GlyR were made to both knees under anaesthesia (100 mg/kg ketamine and 10 mg/kg xylazine, delivered intraperitoneally) when mice were aged 6 weeks.4-weeks later, after capturing baseline behaviours, mice were anaesthetised and one knee (side determined randomly) received an intra-articular injection of 10 µg complete Freund's adjuvant (CFA; Chondrex) to induce inflammation.The width of each knee joint was measured with digital callipers before and 24-hours post-CFA injection.After assessing post-CFA behaviour, mice received an intraperitoneal injection of 0.3 mg/kg varenicline (from a 0.06 mg/ml stock).

Analgesic drugs and recovery scores
Pregabalin (ORB389663-BOR, Stratech) was used as a positive control for tSNI experiments.10 mg/kg was given 1hr prior to von Frey testing on day 14 post SNI.Meloxicam (Metacam, 5 mg/kg; Boehringer Ingelheim) was used as a positive control for knee inflammation experiments.Meloxicam, or saline, mice were tested in a random order each time.Recovery achieved was calculated using the following (Drug data-injury data)/(Baseline data-Injury data), expressed as a percentage.Less than 0 = behavior was worse, 0 = no recovery, 100 = full recovery, more than 100 exceeds full recovery (which would reflect mice being less sensitive that their original baseline).

Behavioural Assays von Frey
Mice were elevated on a wire mesh base in a test box (5 × 5 × 10 cm), and acclimatised to the equipment for 30-60 min.The plantar hind paws were tested using calibrated von Frey hairs (Linton Instrumentation) using the 'up-down' method (Dixson 1980) to evaluate their 50% paw withdrawal thresholds.For spared nerve injury experiments mice were test for mechanical sensitivity over the course of the injury (days 6, 7, 14, 21, 28).

Brush
The plantar hind paws of mice were brushed (1 cm s-1) with a fine artists paint brush.Each mouse received 5 successive stimuli on alternate hind paws (10s apart), twice.The number of responses were recorded.A response included, lifting, flicking or moving the hind paw or walking away from the stimulus.

Pinprick
Noxious mechanosensation was assessed by the pinprick test described previously (83) Mice were housed and acclimatised similarly to the von Frey test.Mice were tested on their plantar hind paws using a sharp pin attached to a 1 g calibrated von Frey filament.Mice were video recorded using a GoPro at 240 fps, and the latency to withdraw from the pinprick analysed by an investigator blind to treatment groups.Three measurements were taken for each hind paw per trial and plotted latency represents the average of both paws.

Hargreaves
Thermal thresholds were assessed using an infrared light source applied to the plantar surface of each hind paw.Three measurements were taken for each hind paw and the averaged latency to withdraw was measured.

Dry Ice
Noxious cold thresholds were measured using the dry ice assay.Mice were elevated on a borosilicate glass (5 mm) platform in a test box.Pieces of dry ice were place into a 2 ml syringe (top cut off).The syringe filled with dry ice was placed against the glass from below (where hind paws were flat and visible).Latency to withdraw paws form the dry ice/glass was measured.Three measurements were taken for each hind paw

Beam task
The Beam test (84) apparatus consisted of a 1-m long horizontal beam of 12-mm width suspended from the ground.
Mice were placed at one end of the beam next to a light source and walked across the beam towards a darkened box/house.Trials were recorded once per day and video footage was used by an investigator blind to treatment groups, to assess number of steps and number of missteps.Baseline data were generated from the average of two trials on separate days. 1 mouse was excluded from the beam test due to abdominal obesity, which prevented the hind paws from being able to reach the beam.

Digging
The digging behaviour of mice was measured as a readout of spontaneous pain.Testing involved placing individual mice into standard 49 × 10 × 12 cm cages filled with ~4 cm tightly packed fine-grain aspen midi wood chip bedding substrate (LBS Biotechnology).Mice were allowed 3 minutes to explore testing cages under video surveillance.
Training sessions were carried out the day before baseline behaviours were captured, during these sessions mice were placed in test cages as per a normal test, however, mice that did not dig for at least 15 seconds were subsequently placed in a test cage with a cage-mate that did meet this criterion until both animals demonstrated digging behaviour.
Following test digs, the number of visible burrows at the end of the 3 minutes was recorded.Digging duration was scored independently by two investigators following the conclusion of each study and blinding of the acquired videos; since the scores of investigators was well correlated (R2 = 0.84, across 174 videos) an average is reported as the digging duration.

Rotarod
Locomotor function and coordination was assessed using a rotarod (Ugo Basile).Mice were placed on the rotarod at a constant speed (7 rpm) for 1 minute before starting an accelerating programme (7-40 rpm, over 5 minutes), test runs were video recorded.Mice were removed from the rotarod if they fell, following two consecutive passive rotations or after 6 minutes of the accelerating program, whichever occurred first.Mice were first placed on the rotarod the day before baseline behaviours were captured to gain some familiarity with the assay.The latency to passive rotation or fall was timed by 1 investigator at the conclusion of each study following blinding of the acquired videos.

Pressure Application Measurement
Mechanical sensitivity of the knee joint was assessed using a pressure application measurement device (Ugo Basile).
Mice received no training in this assay before acquiring baseline sensitivity and digging, and rotarod behaviours were always assessed before the application of pressure to the knee joint.Animals were scruffed before the force transducer was used to apply gradual force to each of the animals' knee joints, by squeezing the joint medially.The withdrawal threshold was recorded when an animal withdrew the limb being tested, or after 450 g force was applied, whichever occurred first.Each animal was tested twice per time point, with a short break between tests, withdrawal force is reported as an average of the two measurements taken at each time point.

Chemical pain model -formalin assay
Mice were chosen at random from their home cage and the left hind paw of each mouse was injected subcutaneously with 2% formalin and mice were immediately placed in a test box (5 x 5 x 10 cm), on a glass base, which was elevated above a camera.The perimeter and roof of the test box consisted of mirrors to allow good visualisation of the injected hindpaw.The mice were video recorded in this environment for 1 hr while the experimenter left the room.Off line analysis was used to measure nocifensive behaviours of the injected hind paw (lifting, licking, flinching, shaking) every 5 mins for 60 mins.The formalin assay was also analysed in 2 phases; 1st phase 0-15 mins, 2nd phase 15-60 mins.All formalin behaviour was conducted 1 hrs post 0.3mg/kg i.p. of varenicline.

Immunohistochemistry
Animals were deeply anaesthetised with pentobarbital and the blood cleared from all tissues by perfusing saline through the vascular system.Mice were then perfuse-fixed using 4% paraformaldehyde (PFA).Tissues were then collected and post-fixed in 4% PFA accordingly (DRG: 1-2 h, spinal cord: 24 h).All tissues were cryoprotected in 30% sucrose for a minimum of 48 h, followed by embedding the tissue and sectioning on a cryostat.(DRG: 12 μm, spinal cord: 15 μm).Cultured cells were fixed with 4% PFA for 10 min and treated similarly to other tissues.Standard immunohistochemistry protocols were used.Briefly, fixed/sectioned samples were washed in PBS and blocked in a blocking solution (5% normal donkey serum, 0.3% TritonX-100, PBS) for 1hr at room temperature (RT).Primary antibodies (Table S1) were diluted in blocking solution and applied to tissue or cells overnight at RT.The next day samples were washed in a wash solution (0.3% TritonX100, PBS) followed by a 2hr incubation with secondary antibodies diluted in wash solution at RT.Samples were mounted using Vectorshield and imaged on a confocal microscope (Zeiss LSM-710).Images were analysed using Fuji/ImageJ (NIH).For quantification at least three sections per animal were used, with at least 3 animals per group.

Fig. S4 .
Fig. S4.Subcutaneous injection of AAV9 in neonatal mice, specifically targets peripheral ganglia, and not spinal neurons.(A) Illustration of experimental and analytical design.Neonatal mice, postnatal days 5-7, received one 10ul subcutaneous injection of AAV9-mCherry at the nape of the neck.4-6 week later tissues (dorsal root ganglia, sympathetic ganglia and spinal cords) were collected and analysed for mCherry expression.(B) Example image of L4 DRG neurons transduced with AAV9-mCherry.Scale bar 100 µm.(C) Quantification of DRG neurons transduced with AAV9-mCherry across cervical, thoracic and lumbar levels.(D) AAV9-mCherry transduced the four major DRG subpopulations.(E) The superior cervical ganglion (sympathetic ganglion) was also transduced by AAV9-mCherry.Scale bar 100 µm.(F) mCherry positive primary afferent terminals can be visualised in the dorsal horn of the spinal cord, following neonatal s.c.injection of AAV9-mCherry.mCherry positive spinal cord neurons were not identified.Scale bar 200 µm.Data mean ± S.E.M.

Fig. S6 .
Fig. S6.Proprioceptive behaviors are preserved following PSAM 4 -GlyR mediated silencing of sensory neurons.(A) Schematic of the experimental design.(B) Depiction of the beam task, mice were trained to walk along a thin beam toward a dark house/space.Steps along the beam were video recorded.(C) Quantification of total number of steps during the beam task.(D) Quantification of the total number of slips or mistakes during the task.(mCherry: n = 10 mice, PSAM 4 -GlyR: n = 11 mice, all data sets RM-two way ANOVA, post-hoc Bonferroni test, P > 0.05).Data expressed as mean ± S.E.M.